Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.
Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.
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